Nearly half of the species of insect feed on plants and these maybe further subdivided into those feeding on green plants (phytophagous) and those feeding on fungi (mycetophagous). Predominantly phytophagous groups of phytophagous insects are: Orthoptera, Lepidoptera, Homoptera, Thysanoptera, Phasmida, Isoptera, Coleoptera.
Primary target pests on monocots (e.g., Cereals, Vegetables and Tubers, Oil Crops, Sugar Crops, Forage and Turf Grasses, Fiber Plants and Woods and Spices and Flavorings) are phytophagous beetles or moths of the orders Coleoptera and Lepidoptera.
Primary target pests on dicots (e.g., dicot selected from the group of plant types consisting of Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops, Forage Legumes, Fiber Plants and Woods and Spices and Flavorings) are also members of the orders Coleoptera and Lepidoptera.
Lepidopteran insects include, but are not limited to, tomato pinworm (Keiferia lycopersicella), tobacco hornworm (Manduca secta), (Trichoplusia ni), and tomato fruitworm (Heliothis zea and Heliothis virescens).
Coleoptera insects include, but are not limited to corn rootworm (Diabrotica spp., such as D. longicornis, D undecimpunctata and D virgifera) and Colorado potato beetle (Leptinotarsa sp., such as L. decemlineata).
Many of such insects are relatively specialized feeders. Simply put, pest resistance may be possible because many pests have specialized to a restricted range of host plant. Because of these specializations and because of continued evolutionary pressure exerted by the multitude of plant secondary substances, the physiological/biochemical systems of the alimentary canals in herbivores and pathogens which are vulnerable to plant chemicals are believed to be distinctive in their chemical properties and distinctive in their susceptibility to disruption. Both the pH and proteases in the insect gut lumen have been suggested as important factors (see Jacquet et al. (1987), Appl. Environ. Microbiol., 53:500-504; and Halder et al. (1986), Eur. J. Biochem, 156:531-540). Consequently, while insects which feed on the same host plant may be expected to contain enzymes having homologous functions, insufficient information is available regarding alimentary canal membranes to provide an expectation as to the similarity of gut membrane tissue between insects, particularly insects of different orders.
The alimentary canal in insects is divided into three main regions: the foregut (stomodaeum), which is ectodermal in origin; the midgut (mesenteron), which is endodermal, and the hindgut (proctodaeum), which is again ectodermal. In many insects these regions are subdivided into various functional parts, of which the most usual are the pharynx, oesophagus, crop and proventriculus in the foregut, the caeca and ventriculus in the midgut, and pylorus, ileum and rectum in the hindgut.
The midgut in the majority of insects is lined by a delicate peritrophic membrane. The peritrophic membrane of insects eating solid food protects the midgut cells from abrasion; the membrane also acts as a barrier to micro-organisms thus reducing infection of the tissues.
The membrane nearly always contains chitin and protein. The most characteristic midgut cells are tall and columnar with regular microvilli forming a striated border adjacent to the lumen. The basal membranes of the cells, adjacent to the haemocoel, are infolded with few openings to the haemolymph so that the extracellular spaces which they enclose are relatively isolated. The division and differentiation of representative cells occur at crypts in the epithelium, which are visible as small papillae on the outside of the midgut. The circular muscles lie adjacent to the epithelium and are bounded by a delicate connective tissue sheath.
Certain bacterial insecticides act very selectively on different kinds of hosts to effectively disrupt processes in cells or enzymes of the alimentary canal.
The most widely used microbial pesticides are derived from the bacterium Bacillus thuringiensis (B. thuringiensis). The spore-forming microorganism Bacillus thuringiensis produces a crystalline protein toxin. The toxin, upon being ingested in its crystalline form by susceptible insect larvae, is transformed into biologically active moieties by the insect gut juice proteases. The primary target is insect cells of the gut epithelium, which are rapidly destroyed.
For example, certain strains of B. thuringiensis produce crystal toxins which are toxic to the larvae of certain lepidopteran insects (see Beegle (1978), Developments in Industrial Microbiology, 20:97-104, and U.S. Pat. No. 4,990,332 to Payne, et. al (Mycogen).
Other strains of B. thuringiensis produce crystal toxins which are effective against certain species of beetles of the order Coleoptera (see 5,017,373 to Herrnstadt et. al. (Mycogen); and see Krieg et al. (1983), Z. ang. Ent., 96:500-508).
The molecular basis for the differences in insecticidal spectrum of B. thuringiensis strains is still incompletely understood. It has been demonstrated that the difference in the insecticidal spectrum of two toxins on the tobacco hornworm (Manduca sexta) and the large white butterfly (Pieris brassicae) was correlated with the presence of high affinity binding sites on the plasma membrane of gut epithelial cells of target insects (see Van Rie et al. (1990), Applied and Environmental Microbiology, 56:1378-1385). Considerable differences in the concentration of binding sites have been observed, reflecting the differences in toxicity (see Hofman et al. (1988), Proc. Natl. Acad. Sci. USA, 85:7844-7848).
Clearly it would be advantagous to have a means for a skilled artisan to target a growth inhibitor agaisnt selected species of insects in both the order Lepidoptera and the order Coleoptera. In some instances a skilled artisan may have a growth inhibitor effective against one or a few species within Lepidoptera or Coleoptera. However, it would may also be desired to target a growth inhibitor against species within Lepidoptera and Coleoptera, thus selectively and simultaneously control insects of two major orders of pests.
As described in more detail below, antibodies are protein tetramers having a binding site for specific binding to an antigenic determinant (epitope). Because of their antigenic specificity, antibodies have been used in various insect studies.
Antibodies to muscles or nerves of the flesh fly Sarcophoga falculata Pand., when fed to the flies, were found to be attached specifically to the tissue and consequently the metabolism of the flies seemed to be severely disturbed (see Schlein et al. (1976), Ann. Trop. Med. Parasitol., 70:227; and Schlein et al. (1976), Physiol. Entomol., 1:55).
Antiserum containing antibodies directed against albumin, a diet constituent, severely disturbed by feeding such flies (see Nogge and Giannetti (1980), Science, 209:1028-1029).
Because of the targeting properties of antibodies and the fact the various flesh eating or blood sucking insects are able to absorb orally administered antibodies. It has been suggested that that it may be possible to use antibodies as an insecticide, providing antigens could be found that gives rise to antibodies that interfere with the metabolism of a target insect.
There are several major concerns associated with the use of antibodies as a selective targeting agent. The antigen against which the antibody is directed must not so unique as to limit the host range to which the antibody may be directed. For example, because of the reported variability of insect alimentary canals, it would not be expected that the midgut epithelium cells of different orders would be suitable targeted antigens. On the other hand, the antigen should not be common so that the antibody is crossreactive to a variety of digestive tissues. For example, it would disadvantageous to have an immunglobulin, particulary if conjugated to a eukaryotic cell inhibitor (e.g., a ribosome inhibiting protein), directed against digestive tissue of insect parasites or predators which normally help control populations of beneficial pests; or directed against digestive tissue of human or other mammal.
To date, no reference has taught the use of a targeting molecule which is capable of recognizing antigenic determinants unique to insects of the orders Lepidoptera and Coleoptera. This lack of a targeting molecule is not surprising because, as previously stated, relatively little is known about invertebrate gut membrane biochemistry--the composition of insect gut membranes in particular have received almost no attention.
Other objects and advantages of the present invention will become apparent from the description of the invention provided hereunder.